EXPRESSION OF HEAT SHOCK GENES IN MOUSE SPERMATOGENIC CELLS

Environmental Health Sciences

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Abstract

The goals of these studies are to determine the regulation of expression and roles of two unique members of the hsp70 heat-shock protein family in male germ cells. The HSP70 proteins are molecular chaperones that assist in the folding of nascent polypeptides and assembly of multimeric complexes, and in the refolding of denatured proteins following heat shock and other stresses. The genes for most HSP70 proteins are expressed constitutively (Hsc70, Hsp78) or in response to stress (Hsp70-1 and Hsp70-3), while Hsp70-2 and Hsc70t are expressed in response to developmental cues and only in male germ cells. Permanent cell lines are not available for promoter analysis studies of gene expression in male germ cells and reliable methods for transfecting primary cells have not been developed. We therefore used transgenic mice to delimit the upstream regulatory region required for developmental expression of Hsp70-2. Different Hsp70-2 gene promoter fragments were ligated to the Lacz reporter gene and beta-galactosidase expression determined in the testes of transgenic mice. It was found that sequences within 604 bp of the translation start site are required for correct expression. This region was examined further with in vitro methods. Footprint analysis identified two domains protected from DNase digestion by germ cell nuclear proteins, referred to as box 1 (between bp ?555 and ?503) and box 2 (between bp ?346 and ?335). These domains contain clusters of transcription factor binding motifs. Gel shift and super-shift analyses indicated that several known transcription factors and unknown proteins present in germ cell nuclei bind to specific sequences in these regions. HSP70-2 is synthesized during the meiotic phase of male germ cell development and we hypothesized that it is a chaperone for proteins involved in meiosis. This was confirmed using the gene knockout approach. Disruption of the Hsp70-2 gene resulted in developmental arrest and apoptosis of pachytene spermatocytes at the G2/M-phase transition of meiosis I. Since this event requires cyclin B1-dependent Cdc2 kinase activity, we further hypothesized that HSP70-2 is a chaperone required for Cdc2 activation. Although Cdc2 was present in the testis of Hsp70-2 knockout mice, it did not form a heterodimer with cyclin B1 and lacked kinase activity. Addition of recombinant HSP70-2 protein to a homogenate of testis from Hsp70-2 knockout mice restored the ability of Cdc2 to form a heterodimer with cyclin B1 and to become an active kinase, confirming that HSP70-2 is a chaperone for Cdc2. However, the effect is leaky and a few germ cells avoid apoptosis, undergo one or sometimes both meiotic divisions and begin acrosome formation. This indicates that some developmental events that normally occur in spermatids do not require completion of meiosis. Similar results occur in Drosophila with mutations in the Cdc25 or Cdc2 genes, suggesting that other kinases may partially compensate in the absence of Cdc2 kinase activity. In addition, heterozygous males have moderately reduced fertility, suggesting a dosage effect of HSP70-2 in germ cell development, and fewer homozygous males are born that expected, suggesting a role for HSP70-2 in sperm function or embryonic development. The HSC70T protein is present only in spermatids, during the post-meiotic phase of male germ cell development. By analogy with HSP70-2, we hypothesized that HSC70T is a chaperone for unique proteins involved in post-meiotic germ cell development. Although male Hsc70t knockout mice have slightly reduced fertility, there are no apparent changes in testis morphology, or sperm numbers and motility. The absence of an overt phenotype suggested that other HSP70 proteins present in spermatids might compensate for the absence of HSC70T, but no changes in mRNA or protein levels were found by northern or western blot analysis. The gene also maps to a region of the MHC on mouse chromosome 17 linked to susceptibility to allergic orchitis. However, analysis of the possible role of HSC70T in this testicular autoimmune disease failed to detect differences between wild-type and Hsc70t knockout mice (collaborator Tung). It remains to be determined if lack of HSC70T increases the sensitivity of spermatids or sperm to the effects of thermal, oxidative, or chemical stresses. Yeast two-hybrid screens identified a unique protein (referred to as protein D1) that binds to HSC70T and HSP70-2 and is expressed predominantly in testis. HSC70T and D1 also bind MSJ-1, a J-domain protein expressed only in post-meiotic germ cells (collaborator Berruti). J-domain proteins bind to a C-terminal region of HSP70 proteins and modulate the specificity of their interactions with other proteins. D1 binds to the N-terminal ATPase domain of HSC70T and HSP70-2 but not HSC70. A recently reported human protein with significant similarity to D1 binds to the death domain on some TNF receptors and was named silencer of death domain (SODD). It also binds HSC70 and was hypothesized to regulate apoptosis by suppressing signal transduction events downstream of death-domain proteins. Current studies are determining if D1modulates HSC70T and MSJ-1 interactions or chaperone activities and the relationship between the D1 and the SODD protein and the role of D1 in germ cell apoptosis will be examined.